This invention relates to inhibition of animal viral replication.
HBV is the prototypic member of the hepadnavirus family, a group of enveloped DNA viruses that primarily infect the liver. HBV infection in humans may lead to significant liver diseases, including acute liver failure, chronic active hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) (McLachlan, Molecular Biology of the Hepatitis B Virus, CRC Press, Boca Raton, Fla., 1991). The HBV genome consists of a partially double-stranded 3.2 kb DNA molecule with a relaxed circular conformation. Sequence analysis of the genome reveals that HBV encodes four partially overlapping open reading frames (ORF) that direct the synthesis of at least seven viral gene products. This expansion of coding capacity is due to the presence of multiple in-frame initiation codons within the precore/core and envelope ORFs generating proteins of different lengths that share a common carboxyl terminus but have different amino terminal regions. This phenomenon is exemplified by the precore/core ORF. The genomic promoter of HBV directs the synthesis of two types of 3.5 kb transcripts that differ at their 5' initiation sites. The shorter transcript initiates 5 nucleotides downstream from the precore initiation codon (Will et al., J. Virol. 61:904-911, 1987).
It serves as the messenger RNA (mRNA) for the 21 kDa core protein and the polymerase (Chang et al., Proc. Natl. Acad. Sci. U.S.A. 87:5158-5162, 1990). Following encapsidation into the viral nucleocapsid, this mRNA species acts as the template for reverse transcription to generate viral DNA (Bartenschlager and Schaller, EMBO J. 11:3413-3420 1992; Hirsch et al., Nature 344:552-555, 1990). The longer transcript, which has a 5'-end extension, directs the synthesis of the precore gene product but the transcript is not packaged into the viral nucleocapsid (Nassal et al., Cell 63:1357-1363, 1990; Yaginuma et al., Proc. Natl. Acad. Sci. U.S.A. 84:2678-2686, 1987).
The 21 kDa viral core protein (p21) assembles into a 180 subunit nucleocapsid structure (Birnbaum and Nassal, J. Virol. 64:3319-3330, 1990; Gallina et al., J. Virol. 63:4645-4652, 1989). This molecule is also involved in nucleic acid binding and promotes viral replication (Hatton et al., J. Virol. 66:5232-5241, 1992; Nassal, J. Virol. 66:4107-4116, 1992).
Translation of the precore mRNA results in a core related polypeptide designated p25. This precore protein is identical to p21 except that it has a 29 amino acid (aa) amino terminal extension. The first 19 aa's of this extension act as a signal peptide, directing the protein into the secretory pathway of the cell (Bruss and Gerlich, Virology 163:268-275, 1988; Ou et al., Proc. Natl. Acad. Sci. U.S.A. 83:1578-1582, 1987). The 19 aa signal peptide is subsequently cleaved to generate a 22 kDa intermediate protein product (p22) that is either translocated to the endoplasmic reticulum (ER) or released back into the cytoplasm (Garcia et al., J. Cell. Biol. 106:1093-1104, 1988). In the ER, p22 is cleaved in an arginine-rich domain near the carboxyl terminus to create a 17 kDa soluble protein (p17) known as HBeAg, which is then secreted from the cell (McLachlan, Molecular Biology of the Hepatitis B Virus, CRC Press, Boca Raton, Fla., 1991). The exact length of p17 is not known, and appears to vary slightly at the carboxyl terminus.
The function of HBeAg in the biology of HBV infection is unknown. HBeAg is found in the serum of HBV-infected individuals, where it generally correlates with high levels of viremia. HBV titers have been found to decrease in serum when there is a detectable anti-HBeAg immune response (McLachlan, Molecular Biology of the Hepatitis B Virus, CRC Press, Boca Raton, Fla., 1991). There is evidence to suggest that HBeAg may function as a circulating protein that blocks cytotoxic T cell activity against HBV core-associated epitopes (Milich et al., Proc.
Natl. Acad. Sci. U.S.A. 87:6599-6603, 1990). Furthermore, it has been demonstrated that HBeAg determinants are expressed on the surface of infected hepatocytes and present HBeAg/HBcAg epitopes in the context of HLA class I molecules to the host immune system (Schlicht and Schaller, J. Virol. 63:5399-5404, 1989).
However, a functional precore gene appears inessential for viral replication, at least in animals experimentally infected with the related duck hepatitis B virus (DHBV) and woodchuck hepatitis virus (WHV) (Chang et al., J. Virol. 61:3322-3325, 1987; Chen et al., J. Virol. 66:5682-5684, 1992; Schlicht et al., J. Virol. 61:3701-3709, 1987). With respect to the human virus HBV, viral genomes defective in HBeAg synthesis are frequently found in individuals with chronic infection. The most common mutation detected is a TGG to TAG transition that introduces an amber termination signal at codon 28 in the precore ORF. This naturally occurring HBV mutant has been associated with fulminant hepatitis and high levels of viral replication (Carman et al., Hepatology 14:219-222, 1991; Liang et al., N. Engl. J. Med. 324:1705-1709, 1991; Omata et al., N. Engl. J. Med. 324:1699-1704, 1991) as well as with chronic infection (Brunetto et al., Ital. J. Gastroenterol. 21:151-154, 1989; Carman et al., Lancet 2:588-591, 1989; Naoumov et al., Gastroenterology 102:538-543, 1992; Okamoto et al., J. Virol. 64:1298-1303, 1990; Tong et al., Virology 176:596-603, 1990). However, it is still unclear if HBeAg minus HBV genomes are associated with a more severe form of chronic liver disease. In this respect, one study demonstrated that transfection of an HBeAg minus genome into human HCC cells resulted in increased viral replicative forms as compared to wild type HBV (Lamberts et al., J. Virol. 67:3756-3762, 1993).